1. Field of the Invention
Methods available for the rapid accurate quantitative or qualitative determination of biologically active substances at extremely low concentrations are limited in number. The physician's diagnosis of the patient or confirmation of the diagnosis frequently involves the detection and/or quantitation of one or more substances in body fluids such as saliva, blood, or urine. The ability to rapidly detect in body fluids the presence and amounts of such materials as may be naturally synthesized by the body or ingested is often times critical to the patient's life. Such materials would include, but are not limited to, hormones of both steroid and polypeptide type, prostaglandins, toxins, and other substances which may be involved in body functions, such as thyroxine, triiodithyronine, etc. The method of assay to be useful to the physician, must be capable of differentiating between extremely small differences in concentrations or amounts of the substance.
2. Description of the Prior Art
Several methods have, in the past, been used for the assay of body fluids, notably radioassay, radioimmunoassay, thin layer chromatography and enzyme amplified assay systems.
The radioimmunoassay procedure has been described by Murphy, Journal, Clinical Endocrinology, 27, 973 (1967); Ibid, 28, 343 (1968). The use of a radioassay or radioimmunoassay technique suffers from several disadvantages among which are the hazards associated with or inherent in radioactive substances, associated handling problems, instability, the need for expensive equipment for the performance of the assays and the difficulties associated with the manipulation and separation of the free and bound forms of the radiolabled substance.
Thin layer chromatography procedures are described by Stahl, Thin Layer Chromatography, Springer Verlag, New York, 1969. The use of methods dependent upon thin layer chromatography for the analysis of trace amounts of materials requires a high degree of proficiency in the performance of the technique, a qualification which limits the usefulness of the method in general. Further, the method is often quite slow in the development of the chromatogram, is sensitive to the presence of a variety of interfering factors and suffers from fluctuations in the range of its performance characteristics or reliability.
An enzyme amplification assay is described by Rubenstein and Ullman, in U.S. Pat. No. 3,819,837. The use of this technique requires the precise and delicate manipulation of biological reagents of extremely complex nature, both with respect to their preparation, storage and usage. Thus, by virtue of their complexity and potential sensitivity to variations in environmental conditions, the enzyme amplification assays have not proven entirely satisfactory with respect to sensitivity and specificity.
It is therefore an object of the present invention to provide an improved method that will detect and accurately determine small amounts of organic substances in body fluids.